Protocol

Molecular Embryology

Volume 97 of the series Methods in Molecular Biology™ pp 601-609

Single-Cell RT-PCR cDNA Subtraction

  • Damian L. WeaverAffiliated withSchool of Biological Sciences, University of Manchester
  • , César NúñezAffiliated withSchool of Biological Sciences, University of Manchester
  • , Clare BrunetAffiliated withSchool of Biological Sciences, University of Manchester
  • , Victoria BostockAffiliated withSchool of Biological Sciences, University of Manchester
  • , Gerard BradyAffiliated withSchool of Biological Sciences, University of Manchester

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Abstract

A major problem in trying to understand complex developmental processes is one of heterogeneity at both the cellular and molecular levels. At the cellular level it is often difficult to identify cells that are undergoing developmental changes and to establish the stage of differentiation they have reached. At the molecular level, there is then a problem in establishing which of the many thousands of expressed genes are playing a role in development. Several approaches to identifying expressed candidate developmental genes are based on comparing the mRNA expression patterns in cells before and after transition points. Differential screening of cDNA libraries with labeled total cDNA probes from contrasting cell samples (1) provides a simple means of identifying genes that are expressed at high levels in one of the samples. cDNA subtraction protocols (2,3) have increased the sensitivity of this type of approach by removing sequences expressed in both samples. The limitation of these approaches lies in the need for large amounts of starting material necessary for cDNA preparation and subtraction. With the advent of the polymerase chain reaction (PCR) (4) and the development of techniques that allow amplification of target sequences expressed in single cells (5), the limitations of applying cDNA subtraction and differential screening have been removed.